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STIM1, an essential and conserved component of store-operated Ca2+ channel function.

Roos J, DiGregorio PJ, Yeromin AV, Ohlsen K, Lioudyno M, Zhang S, Safrina O, Kozak JA, Wagner SL, Cahalan MD, Veliçelebi G, Stauderman KA - J. Cell Biol. (2005)

Bottom Line: RNAi-mediated knockdown of Stim in Drosophila S2 cells significantly reduced TG-dependent Ca2+ entry.Similarly, knockdown of the human homologue STIM1 significantly reduced CRAC channel activity in Jurkat T cells.We propose that STIM1, a ubiquitously expressed protein that is conserved from Drosophila to mammalian cells, plays an essential role in SOC influx and may be a common component of SOC and CRAC channels.

View Article: PubMed Central - PubMed

Affiliation: Torrey Pines Therapeutics, Inc., La Jolla, CA 92037, USA.

ABSTRACT
Store-operated Ca2+ (SOC) channels regulate many cellular processes, but the underlying molecular components are not well defined. Using an RNA interference (RNAi)-based screen to identify genes that alter thapsigargin (TG)-dependent Ca2+ entry, we discovered a required and conserved role of Stim in SOC influx. RNAi-mediated knockdown of Stim in Drosophila S2 cells significantly reduced TG-dependent Ca2+ entry. Patch-clamp recording revealed nearly complete suppression of the Drosophila Ca2+ release-activated Ca2+ (CRAC) current that has biophysical characteristics similar to CRAC current in human T cells. Similarly, knockdown of the human homologue STIM1 significantly reduced CRAC channel activity in Jurkat T cells. RNAi-mediated knockdown of STIM1 inhibited TG- or agonist-dependent Ca2+ entry in HEK293 or SH-SY5Y cells. Conversely, overexpression of STIM1 in HEK293 cells modestly enhanced TG-induced Ca2+ entry. We propose that STIM1, a ubiquitously expressed protein that is conserved from Drosophila to mammalian cells, plays an essential role in SOC influx and may be a common component of SOC and CRAC channels.

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Essential role of gene CG9126 (Stim) in SOC influx in Drosophila S2 cells. (A) Drosophila SOC influx measured in a fluorimeter. Basal-subtracted fluo-4 fluorescence in relative fluorescence units (RFUs) from Drosophila S2 cells in a 96-well plate. Cells were initially in Ca2+-free solution (Ca0). Bars indicate addition of TG (1 μM, solid line) or vehicle (dotted line), followed by 2 mM Ca2+ (Ca2). The TG-independent response can be explained by partial store depletion during exposure to Ca2+-free solution or possible damage to some cells. Traces are averages of recordings from four individual wells. (B) Development of an end point assay for screening gene candidates. Cells were treated as described in A and placed in a fluorimeter 3 min after adding 2 mM Ca2+. Pre-incubating cells with 20 μM 2-APB reduced TG-dependent Ca2+ entry significantly (P < 5 × 10−6: unpaired t test) and to a greater extent than the TG-independent Ca2+ entry (P < 5 × 10−6: unpaired t test); n = 24 for each treatment group. (C) Treatment of Drosophila S2 cells with Stim dsRNA inhibits SOC influx by 90% (P < 10−5; unpaired t test compared with control mock-treated cells). Data represent basal-subtracted RFUs divided by maximal fluorescence (Fmax) to normalize for cell number. The TG-dependent Ca2+ signal can be obtained by subtracting the average TG-independent signal from the Ca2+ signal after treatment with TG. Knockdown of Stim also inhibited the TG-independent Ca2+ signal (vehicle) by <10%, albeit significantly (P < 10−4, unpaired t test). (D) mRNA reduction in Stim dsRNA-treated cells. RNA was isolated from mock-treated cells or cells treated with a dsRNA specific to Stim. RT-PCR analysis was performed using gene-specific primers to Stim or to a control gene, presenilin (PSN). (E) Suppression of other candidate genes did not markedly inhibit TG-induced Ca2+ influx. Cells were mock treated or treated with dsRNA specific for CG11059, CG1560, trp-l, CG8743, or Stim for 5 d. Ca2+ influx was measured after pretreatment with TG. Data represent basal-subtracted RFUs divided by Fmax to normalize for cell number. (F) Knockdown of CG8743 or CG2165 elevates basal intracellular Ca2+ levels. Cells were mock treated (control) or treated with dsRNA specific for CG8743 or CG2165 for 5 d. Data represents basal RFUs divided by the maximum fluorescence, Fmax, to normalize to cell number (P < 0.01, one-way ANOVA, Dunnett's multiple comparison test compared with control mock-treated cells). (B, C, E, and F) Error bars represent means ± SD.
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fig1: Essential role of gene CG9126 (Stim) in SOC influx in Drosophila S2 cells. (A) Drosophila SOC influx measured in a fluorimeter. Basal-subtracted fluo-4 fluorescence in relative fluorescence units (RFUs) from Drosophila S2 cells in a 96-well plate. Cells were initially in Ca2+-free solution (Ca0). Bars indicate addition of TG (1 μM, solid line) or vehicle (dotted line), followed by 2 mM Ca2+ (Ca2). The TG-independent response can be explained by partial store depletion during exposure to Ca2+-free solution or possible damage to some cells. Traces are averages of recordings from four individual wells. (B) Development of an end point assay for screening gene candidates. Cells were treated as described in A and placed in a fluorimeter 3 min after adding 2 mM Ca2+. Pre-incubating cells with 20 μM 2-APB reduced TG-dependent Ca2+ entry significantly (P < 5 × 10−6: unpaired t test) and to a greater extent than the TG-independent Ca2+ entry (P < 5 × 10−6: unpaired t test); n = 24 for each treatment group. (C) Treatment of Drosophila S2 cells with Stim dsRNA inhibits SOC influx by 90% (P < 10−5; unpaired t test compared with control mock-treated cells). Data represent basal-subtracted RFUs divided by maximal fluorescence (Fmax) to normalize for cell number. The TG-dependent Ca2+ signal can be obtained by subtracting the average TG-independent signal from the Ca2+ signal after treatment with TG. Knockdown of Stim also inhibited the TG-independent Ca2+ signal (vehicle) by <10%, albeit significantly (P < 10−4, unpaired t test). (D) mRNA reduction in Stim dsRNA-treated cells. RNA was isolated from mock-treated cells or cells treated with a dsRNA specific to Stim. RT-PCR analysis was performed using gene-specific primers to Stim or to a control gene, presenilin (PSN). (E) Suppression of other candidate genes did not markedly inhibit TG-induced Ca2+ influx. Cells were mock treated or treated with dsRNA specific for CG11059, CG1560, trp-l, CG8743, or Stim for 5 d. Ca2+ influx was measured after pretreatment with TG. Data represent basal-subtracted RFUs divided by Fmax to normalize for cell number. (F) Knockdown of CG8743 or CG2165 elevates basal intracellular Ca2+ levels. Cells were mock treated (control) or treated with dsRNA specific for CG8743 or CG2165 for 5 d. Data represents basal RFUs divided by the maximum fluorescence, Fmax, to normalize to cell number (P < 0.01, one-way ANOVA, Dunnett's multiple comparison test compared with control mock-treated cells). (B, C, E, and F) Error bars represent means ± SD.

Mentions: To examine the role of individual target genes in SOC influx, a 96-well plate fluorescence assay was used to detect changes in intracellular free Ca2+ concentration ([Ca2+]i) evoked by TG in conjunction with RNAi. Selected to include channel-like domains, transmembrane regions, Ca2+-binding domains, or putative function in SOC influx, 170 genes were tested for involvement in Ca2+ signaling by incubating S2 cells with double-stranded RNA (dsRNA) corresponding to 500 bp fragments of the candidate genes (Table S1, available at http://www.jcb.org/cgi/content/full/jcb.200502019/DC1). Control S2 cells displayed both TG-independent and TG-dependent Ca2+ influx (Fig. 1 A). The TG-dependent Ca2+ response reached a plateau within three minutes of re-adding external Ca2+, and was inhibited by 20 μM 2-APB (Fig. 1 B) or 50 μM SKF96365 (not depicted), which is consistent with the block of Drosophila CRAC currents by these compounds (Yeromin et al., 2004). These results suggest that the TG-dependent SOC influx signal reflects activity of the Drosophila CRAC channel. Cells incubated with a dsRNA probe to gene CG9126 (Drosophila Stim) displayed TG-dependent Ca2+ entry that was reduced by >90% compared with control, whereas the TG-independent Ca2+ signal was reduced by only 10% (Fig. 1 C). In parallel, the level of Stim mRNA was reduced by >50% compared with control (Fig. 1 D). A separate dsRNA targeting a different region of Stim (see Materials and methods) produced equivalent suppression of the SOC influx signal (unpublished data). Finally, suppression of Stim did not alter growth rate or loading with fluo-4, which is consistent with healthy cells.


STIM1, an essential and conserved component of store-operated Ca2+ channel function.

Roos J, DiGregorio PJ, Yeromin AV, Ohlsen K, Lioudyno M, Zhang S, Safrina O, Kozak JA, Wagner SL, Cahalan MD, Veliçelebi G, Stauderman KA - J. Cell Biol. (2005)

Essential role of gene CG9126 (Stim) in SOC influx in Drosophila S2 cells. (A) Drosophila SOC influx measured in a fluorimeter. Basal-subtracted fluo-4 fluorescence in relative fluorescence units (RFUs) from Drosophila S2 cells in a 96-well plate. Cells were initially in Ca2+-free solution (Ca0). Bars indicate addition of TG (1 μM, solid line) or vehicle (dotted line), followed by 2 mM Ca2+ (Ca2). The TG-independent response can be explained by partial store depletion during exposure to Ca2+-free solution or possible damage to some cells. Traces are averages of recordings from four individual wells. (B) Development of an end point assay for screening gene candidates. Cells were treated as described in A and placed in a fluorimeter 3 min after adding 2 mM Ca2+. Pre-incubating cells with 20 μM 2-APB reduced TG-dependent Ca2+ entry significantly (P < 5 × 10−6: unpaired t test) and to a greater extent than the TG-independent Ca2+ entry (P < 5 × 10−6: unpaired t test); n = 24 for each treatment group. (C) Treatment of Drosophila S2 cells with Stim dsRNA inhibits SOC influx by 90% (P < 10−5; unpaired t test compared with control mock-treated cells). Data represent basal-subtracted RFUs divided by maximal fluorescence (Fmax) to normalize for cell number. The TG-dependent Ca2+ signal can be obtained by subtracting the average TG-independent signal from the Ca2+ signal after treatment with TG. Knockdown of Stim also inhibited the TG-independent Ca2+ signal (vehicle) by <10%, albeit significantly (P < 10−4, unpaired t test). (D) mRNA reduction in Stim dsRNA-treated cells. RNA was isolated from mock-treated cells or cells treated with a dsRNA specific to Stim. RT-PCR analysis was performed using gene-specific primers to Stim or to a control gene, presenilin (PSN). (E) Suppression of other candidate genes did not markedly inhibit TG-induced Ca2+ influx. Cells were mock treated or treated with dsRNA specific for CG11059, CG1560, trp-l, CG8743, or Stim for 5 d. Ca2+ influx was measured after pretreatment with TG. Data represent basal-subtracted RFUs divided by Fmax to normalize for cell number. (F) Knockdown of CG8743 or CG2165 elevates basal intracellular Ca2+ levels. Cells were mock treated (control) or treated with dsRNA specific for CG8743 or CG2165 for 5 d. Data represents basal RFUs divided by the maximum fluorescence, Fmax, to normalize to cell number (P < 0.01, one-way ANOVA, Dunnett's multiple comparison test compared with control mock-treated cells). (B, C, E, and F) Error bars represent means ± SD.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171946&req=5

fig1: Essential role of gene CG9126 (Stim) in SOC influx in Drosophila S2 cells. (A) Drosophila SOC influx measured in a fluorimeter. Basal-subtracted fluo-4 fluorescence in relative fluorescence units (RFUs) from Drosophila S2 cells in a 96-well plate. Cells were initially in Ca2+-free solution (Ca0). Bars indicate addition of TG (1 μM, solid line) or vehicle (dotted line), followed by 2 mM Ca2+ (Ca2). The TG-independent response can be explained by partial store depletion during exposure to Ca2+-free solution or possible damage to some cells. Traces are averages of recordings from four individual wells. (B) Development of an end point assay for screening gene candidates. Cells were treated as described in A and placed in a fluorimeter 3 min after adding 2 mM Ca2+. Pre-incubating cells with 20 μM 2-APB reduced TG-dependent Ca2+ entry significantly (P < 5 × 10−6: unpaired t test) and to a greater extent than the TG-independent Ca2+ entry (P < 5 × 10−6: unpaired t test); n = 24 for each treatment group. (C) Treatment of Drosophila S2 cells with Stim dsRNA inhibits SOC influx by 90% (P < 10−5; unpaired t test compared with control mock-treated cells). Data represent basal-subtracted RFUs divided by maximal fluorescence (Fmax) to normalize for cell number. The TG-dependent Ca2+ signal can be obtained by subtracting the average TG-independent signal from the Ca2+ signal after treatment with TG. Knockdown of Stim also inhibited the TG-independent Ca2+ signal (vehicle) by <10%, albeit significantly (P < 10−4, unpaired t test). (D) mRNA reduction in Stim dsRNA-treated cells. RNA was isolated from mock-treated cells or cells treated with a dsRNA specific to Stim. RT-PCR analysis was performed using gene-specific primers to Stim or to a control gene, presenilin (PSN). (E) Suppression of other candidate genes did not markedly inhibit TG-induced Ca2+ influx. Cells were mock treated or treated with dsRNA specific for CG11059, CG1560, trp-l, CG8743, or Stim for 5 d. Ca2+ influx was measured after pretreatment with TG. Data represent basal-subtracted RFUs divided by Fmax to normalize for cell number. (F) Knockdown of CG8743 or CG2165 elevates basal intracellular Ca2+ levels. Cells were mock treated (control) or treated with dsRNA specific for CG8743 or CG2165 for 5 d. Data represents basal RFUs divided by the maximum fluorescence, Fmax, to normalize to cell number (P < 0.01, one-way ANOVA, Dunnett's multiple comparison test compared with control mock-treated cells). (B, C, E, and F) Error bars represent means ± SD.
Mentions: To examine the role of individual target genes in SOC influx, a 96-well plate fluorescence assay was used to detect changes in intracellular free Ca2+ concentration ([Ca2+]i) evoked by TG in conjunction with RNAi. Selected to include channel-like domains, transmembrane regions, Ca2+-binding domains, or putative function in SOC influx, 170 genes were tested for involvement in Ca2+ signaling by incubating S2 cells with double-stranded RNA (dsRNA) corresponding to 500 bp fragments of the candidate genes (Table S1, available at http://www.jcb.org/cgi/content/full/jcb.200502019/DC1). Control S2 cells displayed both TG-independent and TG-dependent Ca2+ influx (Fig. 1 A). The TG-dependent Ca2+ response reached a plateau within three minutes of re-adding external Ca2+, and was inhibited by 20 μM 2-APB (Fig. 1 B) or 50 μM SKF96365 (not depicted), which is consistent with the block of Drosophila CRAC currents by these compounds (Yeromin et al., 2004). These results suggest that the TG-dependent SOC influx signal reflects activity of the Drosophila CRAC channel. Cells incubated with a dsRNA probe to gene CG9126 (Drosophila Stim) displayed TG-dependent Ca2+ entry that was reduced by >90% compared with control, whereas the TG-independent Ca2+ signal was reduced by only 10% (Fig. 1 C). In parallel, the level of Stim mRNA was reduced by >50% compared with control (Fig. 1 D). A separate dsRNA targeting a different region of Stim (see Materials and methods) produced equivalent suppression of the SOC influx signal (unpublished data). Finally, suppression of Stim did not alter growth rate or loading with fluo-4, which is consistent with healthy cells.

Bottom Line: RNAi-mediated knockdown of Stim in Drosophila S2 cells significantly reduced TG-dependent Ca2+ entry.Similarly, knockdown of the human homologue STIM1 significantly reduced CRAC channel activity in Jurkat T cells.We propose that STIM1, a ubiquitously expressed protein that is conserved from Drosophila to mammalian cells, plays an essential role in SOC influx and may be a common component of SOC and CRAC channels.

View Article: PubMed Central - PubMed

Affiliation: Torrey Pines Therapeutics, Inc., La Jolla, CA 92037, USA.

ABSTRACT
Store-operated Ca2+ (SOC) channels regulate many cellular processes, but the underlying molecular components are not well defined. Using an RNA interference (RNAi)-based screen to identify genes that alter thapsigargin (TG)-dependent Ca2+ entry, we discovered a required and conserved role of Stim in SOC influx. RNAi-mediated knockdown of Stim in Drosophila S2 cells significantly reduced TG-dependent Ca2+ entry. Patch-clamp recording revealed nearly complete suppression of the Drosophila Ca2+ release-activated Ca2+ (CRAC) current that has biophysical characteristics similar to CRAC current in human T cells. Similarly, knockdown of the human homologue STIM1 significantly reduced CRAC channel activity in Jurkat T cells. RNAi-mediated knockdown of STIM1 inhibited TG- or agonist-dependent Ca2+ entry in HEK293 or SH-SY5Y cells. Conversely, overexpression of STIM1 in HEK293 cells modestly enhanced TG-induced Ca2+ entry. We propose that STIM1, a ubiquitously expressed protein that is conserved from Drosophila to mammalian cells, plays an essential role in SOC influx and may be a common component of SOC and CRAC channels.

Show MeSH
Related in: MedlinePlus